Oligomer-foscarnet conjugates

ABSTRACT

The invention relates to (among other things) oligomer-foscarnet conjugates and related compounds. A conjugate of the invention, when administered by any of a number of administration routes, exhibits advantages over previously administered un-conjugated foscarnet compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 application of InternationalApplication No. PCT/US2009/001561, filed Mar. 12, 2009, designating theUnited States, which claims the benefit of priority under 35 U. S. C.§119(e) to U.S. Provisional Application Ser. No. 61/069,120, filed Mar.12, 2008, both of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

This invention comprises (among other things) chemically modifiedfoscarnets that possess certain advantages over foscarnets lacking thechemical modification. The chemically modified foscarnets describedherein relate to and/or have application(s) in (among others) the fieldsof drug discovery, pharmacotherapy, physiology, organic chemistry andpolymer chemistry.

BACKGROUND OF THE INVENTION

Foscarnet, the tri-sodium salt of phosphonoformic acid, is a potentinhibitor of reverse transcriptase (RT) from human immunodeficiencyvirus type 1 (HIV-1). It acts by selectively inhibiting viral DNApolymerase and reverse transcriptase. It is not phosphorylated into anactive form by viral host cell enzymes. Therefore, it has the advantageof not requiring an activation step before attacking the target viralenzyme. It inhibits reverse transcriptase and is active against HIV andwas approved by the FDA for the treatment of cytomegalovirus (CMV)retinitis in AIDS patients. Foscarnet is also effective in the treatmentof mucocutaneous diseases caused by acyclovir-resistant strains ofherpes simplex virus (HSV) and varicella-zoster virus (VZV) in AIDSpatients.

Foscarnet can only be administrated intravenously because of its verylow oral bioavailability. Although Foscarnet has very good watersolubility, the poor oral absorption is probably due to its triplenegative charge which is an impediment to cellular uptake. Also it isinherently unstable due to the ability of the formic acid moietycontained in the structure to readily decompose into carbon dioxide whenintroduced to an acidic environment, such as the stomach.

A number of research efforts have been devoted to not only improve thestability but the delivery of Foscarnet intracellularly. For example,Hammond et al describe alkylglycerol derivatives of foscarnet(Alkylglycerol prodrugs of phosphonoformate are potent in vitroinhibitors of nucleoside-resistant human immunodeficiency virus type 1and select for resistance mutations that suppress zidovudine resistance.Hammond, J.; Koontz, D.; Bazmi, H.; Beadle, J.; Hostetler, S.; Kini, G.;Aldern, K.; Richman, D.; Hostetler, K.; Mellors, J. Antimicrob. AgentsChemother. 2001, 45, 1621-1628). However, there is a large unmet needfor developing novel foscarnet-like compounds.

The present invention seeks to address these and other needs in the art.

SUMMARY OF THE INVENTION

In one or more embodiments of the invention, a compound is provided, thecompound comprising a foscarnet residue covalently attached via a stableor degradable linkage to a water-soluble, non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

whereinX is a spacer moiety; andPOLY is a water-soluble, non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

whereinX is a spacer moiety; andPOLY is a water-soluble, non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

whereinR² is selected from the group consisting of substituted higher alkyl,unsubstituted higher alkyl, substituted higher alkenyl, unsubstitutedhigher alkenyl, substituted higher alkynyl, and unsubstituted higheralkynyl;X is a spacer moiety; andPOLY is a water-soluble, non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

whereinR² is selected from the group consisting of substituted higher alkyl,unsubstituted higher alkyl, substituted higher alkenyl, unsubstitutedhigher alkenyl, substituted higher alkynyl, and unsubstituted higheralkynyl;X is a spacer moiety; andPOLY is a water-soluble, non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

wherein:each R independently is selected from the group consisting of alkyl,cycloalkyl, alkylamino, aryl, arylamino, heteroaryl, andheterocycloalkyl;R² is selected from the group consisting of substituted higher alkyl,unsubstituted higher alkyl, substituted higher alkenyl, unsubstitutedhigher alkenyl, substituted higher alkynyl, and unsubstituted higheralkynyl;X is a spacer moiety; andPOLY is a water-soluble, non-peptidic oligomer.

The “foscarnet residue” is a compound having a structure of foscarnetthat is altered by the presence of one or more bonds, which bonds serveto attach (either directly or indirectly) one or more water-soluble,non-peptidic oligomers.

In this regard, any foscarnet compound having foscarnet-like activitycan be used as a foscarnet moiety. Exemplary foscarnet moieties have astructure encompassed by Formula I:

In one or more embodiments of the invention, a composition is provided,the composition comprising a compound comprising a foscarnet residuecovalently attached via a stable or degradable linkage to awater-soluble and non-peptidic oligomer, and optionally, apharmaceutically acceptable excipient.

In one or more embodiments of the invention, a dosage form is provided,the dosage form comprising a compound comprising a foscarnet residuecovalently attached via a stable or degradable linkage to awater-soluble, non-peptidic oligomer, wherein the compound is present ina dosage form.

In one or more embodiments of the invention, a method is provided, themethod comprising covalently attaching a water-soluble, non-peptidicoligomer to a foscarnet moiety.

In one or more embodiments of the invention, a method is provided, themethod comprising administering a compound comprising a foscarnetresidue covalently attached via a stable or degradable linkage to awater-soluble, non-peptidic oligomer.

These and other objects, aspects, embodiments and features of theinvention will become more fully apparent to one of ordinary skill inthe art when read in conjunction with the following detaileddescription.

Additional embodiments of the present conjugates, compositions, methods,and the like will be apparent from the following description, examples,and claims. As can be appreciated from the foregoing and followingdescription, each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present disclosure provided that the features included insuch a combination are not mutually inconsistent. In addition, anyfeature or combination of features may be specifically excluded from anyembodiment of the present invention. Additional aspects and advantagesof the present invention are set forth in the following description andclaims, particularly when considered in conjunction with theaccompanying examples and drawings.

BRIEF DESCRIPTION OF THE FIGURES

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DETAILED DESCRIPTION OF THE INVENTION

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“Water soluble, non-peptidic oligomer” indicates an oligomer that is atleast 35% (by weight) soluble, preferably greater than 70% (by weight),and more preferably greater than 95% (by weight) soluble, in water atroom temperature. Typically, an unfiltered aqueous preparation of a“water-soluble” oligomer transmits at least 75%, more preferably atleast 95%, of the amount of light transmitted by the same solution afterfiltering. It is most preferred, however, that the water-solubleoligomer is at least 95% (by weight) soluble in water or completelysoluble in water. With respect to being “non-peptidic,” an oligomer isnon-peptidic when it has less than 35% (by weight) of amino acidresidues.

The terms “monomer,” “monomeric subunit” and “monomeric unit” are usedinterchangeably herein and refer to one of the basic structural units ofa polymer or oligomer. In the case of a homo-oligomer, a singlerepeating structural unit forms the oligomer. In the case of aco-oligomer, two or more structural units are repeated—either in apattern or randomly—to form the oligomer. Preferred oligomers used inconnection with present the invention are homo-oligomers. Thewater-soluble, non-peptidic oligomer typically comprises one or moremonomers serially attached to form a chain of monomers. The oligomer canbe formed from a single monomer type (i.e., is homo-oligomeric) or twoor three monomer types (i.e., is co-oligomeric).

An “oligomer” is a molecule possessing from about 1 to about 30monomers. Specific oligomers for use in the invention include thosehaving a variety of geometries such as linear, branched, or forked, tobe described in greater detail below.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Unless otherwise indicated, a“PEG oligomer” or an oligoethylene glycol is one in which substantiallyall (preferably all) monomeric subunits are ethylene oxide subunits,though, the oligomer may contain distinct end capping moieties orfunctional groups, e.g., for conjugation. PEG oligomers for use in thepresent invention will comprise one of the two following structures:“(CH₂CH₂O)_(n)—” or “—(CH₂C₂O)_(n-1)CH₂CH₂—,” depending upon whether ornot the terminal oxygen(s) has been displaced, e.g., during a synthetictransformation. As stated above, for the PEG oligomers, the variable (n)ranges from about 1 to 30, and the terminal groups and architecture ofthe overall PEG can vary. When PEG further comprises a functional group,A, for linking to, e.g., a small molecule drug, the functional groupwhen covalently attached to a PEG oligomer does not result in formationof (i) an oxygen-oxygen bond (—O—O—, a peroxide linkage), or (ii) anitrogen-oxygen bond (N—O, O—N).

The terms “end-capped” or “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group. Thus, examples ofend-capping moieties include alkoxy (e.g., methoxy, ethoxy andbenzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. In addition, saturated, unsaturated, substituted and unsubstitutedforms of each of the foregoing are envisioned. Moreover, the end-cappinggroup can also be a silane. The end-capping group can alsoadvantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) of interest towhich the polymer is coupled, can be determined by using a suitabledetector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetricmoieties (e.g., dyes), metal ions, radioactive moieties, and the like.Suitable detectors include photometers, films, spectrometers, and thelike. In addition, the end-capping group may contain a targeting moiety.

The term “targeting moiety” is used herein to refer to a molecularstructure that helps the conjugates of the invention to localize to atargeting area, e.g., help enter a cell, or bind a receptor. Preferably,the targeting moiety comprises of vitamin, antibody, antigen, receptor,DNA, RNA, sialyl Lewis X antigen, hyaluronic acid, sugars, cell specificlectins, steroid or steroid derivative, RGD peptide, ligand for a cellsurface receptor, serum component, or combinatorial molecule directedagainst various intra- or extracellular receptors. The targeting moietymay also comprise a lipid or a phospholipid. Exemplary phospholipidsinclude, without limitation, phosphatidylcholines, phospatidylserine,phospatidylinositol, phospatidylglycerol, and phospatidylethanolamine.These lipids may be in the form of micelles or liposomes and the like.The targeting moiety may further comprise a detectable label oralternately a detectable label may serve as a targeting moiety. When theconjugate has a targeting group comprising a detectable label, theamount and/or distribution/location of the polymer and/or the moiety(e.g., active agent) to which the polymer is coupled can be determinedby using a suitable detector. Such labels include, without limitation,fluorescers, chemiluminescers, moieties used in enzyme labeling,colorimetric (e.g., dyes), metal ions, radioactive moieties, goldparticles, and quantum dots.

“Branched,” in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more polymer “arms”extending from a branch point.

“Forked,” in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more functional groups(typically through one or more atoms) extending from a branch point.

A “branch point” refers to a bifurcation point comprising one or moreatoms at which an oligomer branches or forks from a linear structureinto one or more additional arms.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive,” with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions that are effective to produce a desiredreaction in the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group may vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof encompasses protected forms thereof.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively labile bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater may depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include butare not limited to carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides,oligonucleotides, thioesters, and carbonates.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “stable” linkage or bond refers to a chemical bond that issubstantially stable in water, that is to say, does not undergohydrolysis under physiological conditions to any appreciable extent overan extended period of time. Examples of hydrolytically stable linkagesinclude but are not limited to the following: carbon-carbon bonds (e.g.,in aliphatic chains), ethers, amides, urethanes, amines, and the like.Generally, a stable linkage is one that exhibits a rate of hydrolysis ofless than about 1-2% per day under physiological conditions. Hydrolysisrates of representative chemical bonds can be found in most standardchemistry textbooks.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater, more preferably 97% or greater, still morepreferably 98% or greater, even more preferably 99% or greater, yetstill more preferably 99.9% or greater, with 99.99% or greater beingmost preferred of some given quantity.

“Monodisperse” refers to an oligomer composition wherein substantiallyall of the oligomers in the composition have a well-defined, singlemolecular weight and defined number of monomers, as determined bychromatography or mass spectrometry. Monodisperse oligomer compositionsare in one sense pure, that is, substantially having a single anddefinable number (as a whole number) of monomers rather than a largedistribution. A monodisperse oligomer composition possesses a MW/Mnvalue of 1.0005 or less, and more preferably, a MW/Mn value of 1.0000.By extension, a composition comprised of monodisperse conjugates meansthat substantially all oligomers of all conjugates in the compositionhave a single and definable number (as a whole number) of monomersrather than a large distribution and would possess a MW/Mn value of1.0005, and more preferably, a MW/Mn value of 1.0000 if the oligomerwere not attached to the therapeutic moiety. A composition comprised ofmonodisperse conjugates may, however, include one or more nonconjugatesubstances such as solvents, reagents, excipients, and so forth.

“Bimodal,” in reference to an oligomer composition, refers to anoligomer composition wherein substantially all oligomers in thecomposition have one of two definable and different numbers (as wholenumbers) of monomers rather than a large distribution, and whosedistribution of molecular weights, when plotted as a number fractionversus molecular weight, appears as two separate identifiable peaks.Preferably, for a bimodal oligomer composition as described herein, eachpeak is generally symmetric about its mean, although the size of the twopeaks may differ. Ideally, the polydispersity index of each peak in thebimodal distribution, Mw/Mn, is 1.01 or less, more preferably 1.001 orless, and even more preferably 1.0005 or less, and most preferably aMW/Mn value of 1.0000. By extension, a composition comprised of bimodalconjugates means that substantially all oligomers of all conjugates inthe composition have one of two definable and different numbers (aswhole numbers) of monomers rather than a large distribution and wouldpossess a MW/Mn value of 1.01 or less, more preferably 1.001 or less andeven more preferably 1.0005 or less, and most preferably a MW/Mn valueof 1.0000 if the oligomer were not attached to the therapeutic moiety. Acomposition comprised of bimodal conjugates may, however, include one ormore nonconjugate substances such as solvents, reagents, excipients, andso forth.

A “foscarnet moiety” refers to an organic, inorganic, or organometalliccompound typically having a molecular weight of less than about 1000Daltons (and typically less than 500 Daltons) and having some degree ofactivity as foscarnet-like activity.

A “biological membrane” is any membrane made of cells or tissues thatserves as a barrier to at least some foreign entities or otherwiseundesirable materials. As used herein a “biological membrane” includesthose membranes that are associated with physiological protectivebarriers including, for example: the blood-brain barrier (BBB); theblood-cerebrospinal fluid barrier; the blood-placental barrier; theblood-milk barrier; the blood-testes barrier; and mucosal barriersincluding the vaginal mucosa, urethral mucosa, anal mucosa, buccalmucosa, sublingual mucosa, and rectal mucosa. Unless the context clearlydictates otherwise, the term “biological membrane” does not includethose membranes associated with the middle gastro-intestinal tract(e.g., stomach and small intestines).

A “biological membrane crossing rate,” provides a measure of acompound's ability to cross a biological membrane, such as theblood-brain barrier (“BBB”). A variety of methods may be used to assesstransport of a molecule across any given biological membrane. Methods toassess the biological membrane crossing rate associated with any givenbiological barrier (e.g., the blood-cerebrospinal fluid barrier, theblood-placental barrier, the blood-milk barrier, the intestinal barrier,and so forth), are known, described herein and/or in the relevantliterature, and/or may be determined by one of ordinary skill in theart.

A “reduced rate of metabolism” refers to a measurable reduction in therate of metabolism of a water-soluble oligomer-small molecule drugconjugate as compared to the rate of metabolism of the small moleculedrug not attached to the water-soluble oligomer (i.e., the smallmolecule drug itself) or a reference standard material. In the specialcase of “reduced first pass rate of metabolism,” the same “reduced rateof metabolism” is required except that the small molecule drug (orreference standard material) and the corresponding conjugate areadministered orally. Orally administered drugs are absorbed from thegastro-intestinal tract into the portal circulation and may pass throughthe liver prior to reaching the systemic circulation. Because the liveris the primary site of drug metabolism or biotransformation, asubstantial amount of drug may be metabolized before it ever reaches thesystemic circulation. The degree of first pass metabolism, and thus, anyreduction thereof, may be measured by a number of different approaches.For instance, animal blood samples may be collected at timed intervalsand the plasma or serum analyzed by liquid chromatography/massspectrometry for metabolite levels. Other techniques for measuring a“reduced rate of metabolism” associated with the first pass metabolismand other metabolic processes are known, described herein and/or in therelevant literature, and/or may be determined by one of ordinary skillin the art. Preferably, a conjugate of the invention may provide areduced rate of metabolism reduction satisfying at least one of thefollowing values: at least about 30%; at least about 40%; at least about50%; at least about 60%; at least about 70%; at least about 80%; and atleast about 90%. A compound (such as a small molecule drug or conjugatethereof) that is “orally bioavailable” is one that preferably possessesa bioavailability when administered orally of greater than 25%, andpreferably greater than 70%, where a compound's bioavailability is thefraction of administered drug that reaches the systemic circulation inunmetabolized form.

“Alkyl” refers to a hydrocarbon chain, ranging from about 1 to 20 atomsin length. Such hydrocarbon chains are preferably but not necessarilysaturated and may be branched or straight chain. Exemplary alkyl groupsinclude methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl,2-ethylpropyl, 3-methylpentyl, and the like. As used herein, “alkyl”includes cycloalkyl when three or more carbon atoms are referenced. An“alkenyl” group is an alkyl of 2 to 20 carbon atoms with at least onecarbon-carbon double bond.

The terms “substituted alkyl” or “substituted C_(q-r) alkyl” where q andr are integers identifying the range of carbon atoms contained in thealkyl group, denotes the above alkyl groups that are substituted by one,two or three halo (e.g., F, Cl, Br, I), trifluoromethyl, hydroxy, C₁₋₇alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, and soforth), C₁₋₇ alkoxy, C₁₋₇ acyloxy, C₃₋₇ heterocyclic, amino, phenoxy,nitro, carboxy, acyl, cyano. The substituted alkyl groups may besubstituted once, twice or three times with the same or with differentsubstituents.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl. “Lower alkenyl” refers to a loweralkyl group of 2 to 6 carbon atoms having at least one carbon-carbondouble bond.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy, etc.),preferably C₁-C₇.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to component that may be included in the compositions ofthe invention causes no significant adverse toxicological effects to apatient.

The term “aryl” means an aromatic group having up to 14 carbon atoms.Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl,naphthalenyl, and the like. “Substituted phenyl” and “substituted aryl”denote a phenyl group and aryl group, respectively, substituted withone, two, three, four or five (e.g. 1-2, 1-3 or 1-4 substituents) chosenfrom halo (F, Cl, Br, I), hydroxy, cyano, nitro, alkyl (e.g., C₁₋₆alkyl), alkoxy (e.g., C₁₋₆ alkoxy), benzyloxy, carboxy, aryl, and soforth.

Chemical moieties are defined and referred to throughout primarily asunivalent chemical moieties (e.g., alkyl, aryl, etc.). Nevertheless,such terms are also used to convey corresponding multivalent moietiesunder the appropriate structural circumstances clear to those skilled inthe art. For example, while an “alkyl” moiety generally refers to amonovalent radical (e.g., CH₃—CH₂—), in certain circumstances a bivalentlinking moiety can be “alkyl,” in which case those skilled in the artwill understand the alkyl to be a divalent radical (e.g., —CH₂—CH₂—),which is equivalent to the term “alkylene.” (Similarly, in circumstancesin which a divalent moiety is required and is stated as being “aryl,”those skilled in the art will understand that the term “aryl” refers tothe corresponding multivalent moiety, arylene). All atoms are understoodto have their normal number of valences for bond formation (i.e., 1 forH, 4 for carbon, 3 for N, 2 for 0, and 2, 4, or 6 for S, depending onthe oxidation state of the S).

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a water-soluble oligomer-small moleculedrug conjugate present in a composition that is needed to provide adesired level of active agent and/or conjugate in the bloodstream or inthe target tissue. The precise amount may depend upon numerous factors,e.g., the particular active agent, the components and physicalcharacteristics of the composition, intended patient population, patientconsiderations, and may readily be determined by one skilled in the art,based upon the information provided herein and available in the relevantliterature.

A “difunctional” oligomer is an oligomer having two functional groupscontained therein, typically at its termini. When the functional groupsare the same, the oligomer is said to be homodifunctional. When thefunctional groups are different, the oligomer is said to beheterodifunctional.

A basic reactant or an acidic reactant described herein include neutral,charged, and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as described herein, and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may but need not necessarily occur, so that the descriptionincludes instances where the circumstance occurs and instances where itdoes not.

As indicated above, the present invention is directed to (among otherthings) a compound comprising a foscarnet residue covalently attachedvia a stable or degradable linkage to a water-soluble, non-peptidicoligomer.

The “foscarnet residue” is a compound having a structure of a foscarnetcompound that is altered by the presence of one or more bonds, whichbonds serve to attach (either directly or indirectly) one or morewater-soluble, non-peptidic oligomers. Exemplary foscarnets have astructure defined herein as Formula I:

In some instances, foscarnets can be obtained from commercial sources,e.g., Sigma Chemical CO. St. Louis, Mo.). In addition, foscarnets can beobtained through chemical synthesis. Examples of foscarnets as well assynthetic approaches for preparing foscarnets are described in theliterature and in, for example, U.S. Pat. No. 4,215,113. Each of these(and other) foscarnets can be covalently attached (either directly orthrough one or more atoms) to a water-soluble and non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

whereinX is a spacer moiety; andPOLY is a water-soluble, non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

whereinX is a spacer moiety; andPOLY is a water-soluble, non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

whereinR² is selected from the group consisting of substituted higher alkyl,unsubstituted higher alkyl, substituted higher alkenyl, unsubstitutedhigher alkenyl, substituted higher alkynyl, and unsubstituted higheralkynyl;X is a spacer moiety; andPOLY is a water-soluble, non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

whereinR² is selected from the group consisting of substituted higher alkyl,unsubstituted higher alkyl, substituted higher alkenyl, unsubstitutedhigher alkenyl, substituted higher alkynyl, and unsubstituted higheralkynyl;X is a spacer moiety; andPOLY is a water-soluble, non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

wherein:each R independently is selected from the group consisting of alkyl,cycloalkyl, alkylamino, aryl, arylamino, heteroaryl, andheterocycloalkyl;R² is selected from the group consisting of substituted higher alkyl,unsubstituted higher alkyl, substituted higher alkenyl, unsubstitutedhigher alkenyl, substituted higher alkynyl, and unsubstituted higheralkynyl;X is a spacer moiety; andPOLY is a water-soluble, non-peptidic oligomer.

Use of oligomers (e.g., from a monodisperse or bimodal composition ofoligomers, in contrast to relatively impure compositions) to formoligomer-containing compounds may advantageously alter certainproperties associated with the corresponding small molecule drug. Forinstance, a compound of the invention, when administered by any of anumber of suitable administration routes, such as parenteral, oral,transdermal, buccal, pulmonary, or nasal, exhibits reduced penetrationacross the blood-brain barrier. It is preferred that the compounds ofthe invention exhibit slowed, minimal or effectively no crossing of theblood-brain barrier, while still crossing the gastro-intestinal (GI)walls and into the systemic circulation if oral delivery is intended.Moreover, the compounds of the invention maintain a degree ofbioactivity as well as bioavailability in comparison to the bioactivityand bioavailability of the compound free of all oligomers.

With respect to the blood-brain barrier (“BBB”), this barrier restrictsthe transport of drugs from the blood to the brain. This barrierconsists of a continuous layer of unique endothelial cells joined bytight junctions. The cerebral capillaries, which comprise more than 95%of the total surface area of the BBB, represent the principal route forthe entry of most solutes and drugs into the central nervous system.

For compounds whose degree of blood-brain barrier crossing ability isnot readily known, such ability may be determined using a suitableanimal model such as an in situ rat brain perfusion (“RBP”) model asdescribed herein. Briefly, the RBP technique involves cannulation of thecarotid artery followed by perfusion with a compound solution undercontrolled conditions, followed by a wash out phase to remove compoundremaining in the vascular space. (Such analyses may be conducted, forexample, by contract research organizations such as Absorption Systems,Exton, Pa.). In one example of the RBP model, a cannula is placed in theleft carotid artery and the side branches are tied off. A physiologicbuffer containing the analyte (typically but not necessarily at a 5micromolar concentration level) is perfused at a flow rate of about 10mL/minute in a single pass perfusion experiment. After 30 seconds, theperfusion is stopped and the brain vascular contents are washed out withcompound-free buffer for an additional 30 seconds. The brain tissue isthen removed and analyzed for compound concentrations via liquidchromatograph with tandem mass spectrometry detection (LC/MS/MS).Alternatively, blood-brain barrier permeability can be estimated basedupon a calculation of the compound's molecular polar surface area(“PSA”), which is defined as the sum of surface contributions of polaratoms (usually oxygens, nitrogens and attached hydrogens) in a molecule.The PSA has been shown to correlate with compound transport propertiessuch as blood-brain barrier transport. Methods for determining acompound's PSA can be found, e.g., in, Ertl, P., et al., J. Med. Chem.2000, 43, 3714-3717; and Kelder, J., et al., Pharm. Res. 1999, 16,1514-1519.

With respect to the blood-brain barrier, the water-soluble, non-peptidicoligomer-small molecule drug conjugate exhibits a blood-brain barriercrossing rate that is reduced as compared to the crossing rate of thesmall molecule drug not attached to the water-soluble, non-peptidicoligomer. Exemplary reductions in blood-brain barrier crossing rates forthe compounds described herein include reductions of: at least about 5%;at least about 10%; at least about 25%; at least about 30%; at leastabout 40%; at least about 50%; at least about 60%; at least about 70%;at least about 80%; or at least about 90%, when compared to theblood-brain barrier crossing rate of the small molecule drug notattached to the water-soluble oligomer. A preferred reduction in theblood-brain barrier crossing rate for a conjugate of the invention is atleast about 20%.

Assays for determining whether a given compound (regardless of whetherthe compound includes a water-soluble, non-peptidic oligomer or not) canact as a foscarnet are known and/or may be prepared by one of ordinaryskill in the art and are further described infra.

Each of these (and other) foscarnet moieties can be covalently attached(either directly or through one or more atoms) to a water-soluble andnon-peptidic oligomer.

Exemplary molecular weights of small molecule drugs include molecularweights of: less than about 950; less than about 900; less than about850; less than about 800; less than about 750; less than about 700; lessthan about 650; less than about 600; less than about 550; less thanabout 500; less than about 450; less than about 400; less than about350; and less than about 300 Daltons.

The small molecule drug used in the invention, if chiral, may beobtained from a racemic mixture, or an optically active form, forexample, a single optically active enantiomer, or any combination orratio of enantiomers (i.e., scalemic mixture). In addition, the smallmolecule drug may possess one or more geometric isomers. With respect togeometric isomers, a composition can comprise a single geometric isomeror a mixture of two or more geometric isomers. A small molecule drug foruse in the present invention can be in its customary active form, or maypossess some degree of modification. For example, a small molecule drugmay have a targeting agent, tag, or transporter attached thereto, priorto or after covalent attachment of an oligomer. Alternatively, the smallmolecule drug may possess a lipophilic moiety attached thereto, such asa phospholipid (e.g., distearoylphosphatidylethanolamine or “DSPE,”dipalmitoylphosphatidylethanolamine or “DPPE,” and so forth) or a smallfatty acid. In some instances, however, it is preferred that the smallmolecule drug moiety does not include attachment to a lipophilic moiety.

The foscarnet moiety for coupling to a water-soluble, non-peptidicoligomer possesses a free hydroxyl, carboxyl, thio, amino group, or thelike (i.e., “handle”) suitable for covalent attachment to the oligomer.In addition, the foscarnet moiety may be modified by introduction of areactive group, preferably by conversion of one of its existingfunctional groups to a functional group suitable for formation of astable covalent linkage between the oligomer and the drug.

Accordingly, each oligomer is composed of up to three different monomertypes selected from the group consisting of: alkylene oxide, such asethylene oxide or propylene oxide; olefinic alcohol, such as vinylalcohol, 1-propenol or 2-propenol; vinyl pyrrolidone; hydroxyalkylmethacrylamide or hydroxyalkyl methacrylate, where alkyl is preferablymethyl; α-hydroxy acid, such as lactic acid or glycolic acid;phosphazene, oxazoline, amino acids, carbohydrates such asmonosaccharides, alditol such as mannitol; and N-acryloylmorpholine.Preferred monomer types include alkylene oxide, olefinic alcohol,hydroxyalkyl methacrylamide or methacrylate, N-acryloylmorpholine, andα-hydroxy acid. Preferably, each oligomer is, independently, aco-oligomer of two monomer types selected from this group, or, morepreferably, is a homo-oligomer of one monomer type selected from thisgroup.

The two monomer types in a co-oligomer may be of the same monomer type,for example, two alkylene oxides, such as ethylene oxide and propyleneoxide. Preferably, the oligomer is a homo-oligomer of ethylene oxide.Usually, although not necessarily, the terminus (or termini) of theoligomer that is not covalently attached to a small molecule is cappedto render it unreactive. Alternatively, the terminus may include areactive group. When the terminus is a reactive group, the reactivegroup is either selected such that it is unreactive under the conditionsof formation of the final oligomer or during covalent attachment of theoligomer to a small molecule drug, or it is protected as necessary. Onecommon end-functional group is hydroxyl or —OH, particularly foroligoethylene oxides.

The water-soluble, non-peptidic oligomer (e.g., “POLY” in variousstructures provided herein) can have any of a number of differentgeometries. For example, the water-soluble, non-peptidic oligomer may belinear, branched, or forked. Most typically, the water-soluble,non-peptidic oligomer is linear or is branched, for example, having onebranch point. Although much of the discussion herein is focused uponpoly(ethylene oxide) as an illustrative oligomer, the discussion andstructures presented herein can be readily extended to encompass anywater-soluble, non-peptidic oligomers described above.

The molecular weight of the water-soluble, non-peptidic oligomer,excluding the linker portion, is generally relatively low. Exemplaryvalues of the molecular weight of the water-soluble polymer include:below about 1500; below about 1450; below about 1400; below about 1350;below about 1300; below about 1250; below about 1200; below about 1150;below about 1100; below about 1050; below about 1000; below about 950;below about 900; below about 850; below about 800; below about 750;below about 700; below about 650; below about 600; below about 550;below about 500; below about 450; below about 400; below about 350;below about 300; below about 250; below about 200; and below about 100Daltons.

Exemplary ranges of molecular weights of the water-soluble, non-peptidicoligomer (excluding the linker) include: from about 100 to about 1400Daltons; from about 100 to about 1200 Daltons; from about 100 to about800 Daltons; from about 100 to about 500 Daltons; from about 100 toabout 400 Daltons; from about 200 to about 500 Daltons; from about 200to about 400 Daltons; from about 75 to 1000 Daltons; and from about 75to about 750 Daltons.

Preferably, the number of monomers in the water-soluble, non-peptidicoligomer falls within one or more of the following ranges: between about1 and about 30 (inclusive); between about 1 and about 25; between about1 and about 20; between about 1 and about 15; between about 1 and about12; between about 1 and about 10. In certain instances, the number ofmonomers in series in the oligomer (and the corresponding conjugate) isone of 1, 2, 3, 4, 5, 6, 7, or 8. In additional embodiments, theoligomer (and the corresponding conjugate) contains 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 monomers. In yet further embodiments, theoligomer (and the corresponding conjugate) possesses 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 monomers in series. Thus, for example, when thewater-soluble and non-peptidic polymer includes CH₃—(OCH₂CH₂)_(n)—, “n”is an integer that can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, andcan fall within one or more of the following ranges: between about 1 andabout 25; between about 1 and about 20; between about 1 and about 15;between about 1 and about 12; between about 1 and about 10.

When the water-soluble, non-peptidic oligomer has 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 monomers, these values correspond to a methoxy end-cappedoligo(ethylene oxide) having a molecular weights of about 75, 119, 163,207, 251, 295, 339, 383, 427, and 471 Daltons, respectively. When theoligomer has 11, 12, 13, 14, or 15 monomers, these values correspond tomethoxy end-capped oligo(ethylene oxide) having molecular weightscorresponding to about 515, 559, 603, 647, and 691 Daltons,respectively.

When the water-soluble, non-peptidic oligomer is attached to thefoscarnet (in contrast to the step-wise addition of one or more monomersto effectively “grow” the oligomer onto the foscarnet), it is preferredthat the composition containing an activated form of the water-soluble,non-peptidic oligomer be monodisperse. In those instances, however,where a bimodal composition is employed, the composition will possess abimodal distribution centering around any two of the above numbers ofmonomers. For instance, a bimodal oligomer may have any one of thefollowing exemplary combinations of monomer subunits: 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, and so forth; 2-3, 2-4, 2-5, 2-6, 2-7,2-8, 2-9, 2-10, and so forth; 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, and soforth; 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, and so forth; 5-6, 5-7, 5-8, 5-9,5-10, and so forth; 6-7, 6-8, 6-9, 6-10, and so forth; 7-8, 7-9, 7-10,and so forth; and 8-9, 8-10, and so forth.

In some instances, the composition containing an activated form of thewater-soluble, non-peptidic oligomer will be trimodal or eventetramodal, possessing a range of monomers units as previouslydescribed. Oligomer compositions possessing a well-defined mixture ofoligomers (i.e., being bimodal, trimodal, tetramodal, and so forth) canbe prepared by mixing purified monodisperse oligomers to obtain adesired profile of oligomers (a mixture of two oligomers differing onlyin the number of monomers is bimodal; a mixture of three oligomersdiffering only in the number of monomers is trimodal; a mixture of fouroligomers differing only in the number of monomers is tetramodal), oralternatively, can be obtained from column chromatography of apolydisperse oligomer by recovering the “center cut”, to obtain amixture of oligomers in a desired and defined molecular weight range.

It is preferred that the water-soluble, non-peptidic oligomer isobtained from a composition that is preferably unimolecular ormonodisperse. That is, the oligomers in the composition possess the samediscrete molecular weight value rather than a distribution of molecularweights. Some monodisperse oligomers can be purchased from commercialsources such as those available from Sigma-Aldrich, or alternatively,can be prepared directly from commercially available starting materialssuch as Sigma-Aldrich. Water-soluble, non-peptidic oligomers can beprepared as described in Chen Y., Baker, G. L., J. Org. Chem., 6870-6873(1999), WO 02/098949, and U.S. Patent Application Publication2005/0136031.

When present, the spacer moiety (through which the water-soluble,non-peptidic polymer is attached to the foscarnet moiety) may be asingle bond, a single atom, such as an oxygen atom or a sulfur atom, twoatoms, or a number of atoms. A spacer moiety is typically but is notnecessarily linear in nature. The spacer moiety, “X,” is hydrolyticallystable, and is preferably also enzymatically stable. Preferably, thespacer moiety “X” is one having a chain length of less than about 12atoms, and preferably less than about 10 atoms, and even more preferablyless than about 8 atoms and even more preferably less than about 5atoms, whereby length is meant the number of atoms in a single chain,not counting substituents. For instance, a urea linkage such as this,R_(oligomer)—NH—(C═O)—NH—R′_(drug), is considered to have a chain lengthof 3 atoms (—NH—C(O)—NH—). In selected embodiments, the linkage does notcomprise further spacer groups.

In some instances, the spacer moiety “X” comprises an ether, amide,urethane, amine, thioether, urea, or a carbon-carbon bond. Functionalgroups such as those discussed below, and illustrated in the examples,are typically used for forming the linkages. The spacer moiety may lesspreferably also comprise (or be adjacent to or flanked by) other atoms,as described further below.

More specifically, in selected embodiments, a spacer moiety of theinvention, X, may be any of the following: “—” (i.e., a covalent bond,that may be stable or degradable, between the foscarnet residue and thewater-soluble, non-peptidic oligomer), —O—, —NH—, —S—, —C(O)—, —C(O)O—,—OC(O)—, —CH₂—C(O)O—, —CH₂—OC(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, C(O)—NH,NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—,—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH —, —NH—C(O)—CH₂—, —CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—, —CH₂—NH—C(O)—CH₂—CH₂,—CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—,—CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—,—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—,—CH₂—CH₂—C(O)—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group,—N(R⁶)—, R⁶ is H or an organic radical selected from the groupconsisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl and substituted aryl. Additionalspacer moieties include, acylamino, acyl, aryloxy, alkylene bridgecontaining between 1 and 5 inclusive carbon atoms, alkylamino,dialkylamino having about 2 to 4 inclusive carbon atoms, piperidino,pyrrolidino, N-(lower alkyl)-2-piperidyl, morpholino, 1-piperizinyl,4-(lower alkyl)-1-piperizinyl, 4-(hydroxyl-lower alkyl)-1-piperizinyl,4-(methoxy-lower alkyl)-1-piperizinyl, and guanidine. In some instances,a portion or a functional group of the drug compound may be modified orremoved altogether to facilitate attachment of the oligomer. In someinstances, it is preferred that X is not an amide, i.e., —CONR— or—RNCO—).

For purposes of the present invention, however, a group of atoms is notconsidered a linkage when it is immediately adjacent to an oligomersegment, and the group of atoms is the same as a monomer of the oligomersuch that the group would represent a mere extension of the oligomerchain.

The linkage “X” between the water-soluble, non-peptidic oligomer and thesmall molecule is typically formed by reaction of a functional group ona terminus of the oligomer (or nascent oligomer when it is desired to“grow” the oligomer onto the foscarnet) with a corresponding functionalgroup within the foscarnet. Illustrative reactions are described brieflybelow. For example, an amino group on an oligomer may be reacted with acarboxylic acid or an activated carboxylic acid derivative on the smallmolecule, or vice versa, to produce an amide linkage. Alternatively,reaction of an amine on an oligomer with an activated carbonate (e.g.succinimidyl or benzotriazolyl carbonate) on the drug, or vice versa,forms a carbamate linkage. Reaction of an amine on an oligomer with anisocyanate (R—N═C═O) on a drug, or vice versa, forms a urea linkage(R—NH—(C═O)—NH—R′). Further, reaction of an alcohol (alkoxide) group onan oligomer with an alkyl halide, or halide group within a drug, or viceversa, forms an ether linkage. In yet another coupling approach, a smallmolecule having an aldehyde function is coupled to an oligomer aminogroup by reductive amination, resulting in formation of a secondaryamine linkage between the oligomer and the small molecule.

A particularly preferred water-soluble, non-peptidic oligomer is anoligomer bearing an aldehyde functional group. In this regard, theoligomer will have the following structure:CH₃O—(CH₂—CH₂—O)_(n)—(CH₂)_(p)—C(O)H, wherein (n) is one of 1, 2, 3, 4,5, 6, 7, 8, 9 and 10 and (p) is one of 1, 2, 3, 4, 5, 6 and 7. Preferred(n) values include 3, 5 and 7 and preferred (p) values 2, 3 and 4.

The termini of the water-soluble, non-peptidic oligomer not bearing afunctional group may be capped to render it unreactive. When theoligomer includes a further functional group at a terminus other thanthat intended for formation of a conjugate, that group is eitherselected such that it is unreactive under the conditions of formation ofthe linkage “X,” or it is protected during the formation of the linkage“X.”

As stated above, the water-soluble, non-peptidic oligomer includes atleast one functional group prior to conjugation. The functional grouptypically comprises an electrophilic or nucleophilic group for covalentattachment to a small molecule, depending upon the reactive groupcontained within or introduced into the small molecule. Examples ofnucleophilic groups that may be present in either the oligomer or thesmall molecule include hydroxyl, amine, hydrazine (—NHNH₂), hydrazide(—C(O)NHNH₂), and thiol. Preferred nucleophiles include amine,hydrazine, hydrazide, and thiol, particularly amine. Most small moleculedrugs for covalent attachment to an oligomer will possess a freehydroxyl, amino, thio, aldehyde, ketone, or carboxyl group.

Examples of electrophilic functional groups that may be present ineither the oligomer or the small molecule include carboxylic acid,carboxylic ester, particularly imide esters, orthoester, carbonate,isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate,methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy,sulfonate, thiosulfonate, silane, alkoxysilane, and halosilane. Morespecific examples of these groups include succinimidyl ester orcarbonate, imidazoyl ester or carbonate, benzotriazole ester orcarbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyldisulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, andtresylate (2,2,2-trifluoroethanesulfonate).

Also included are sulfur analogs of several of these groups, such asthione, thione hydrate, thioketal, 2-thiazolidine thione, etc., as wellas hydrates or protected derivatives of any of the above moieties (e.g.aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, ketal,thioketal, thioacetal).

An “activated derivative” of a carboxylic acid refers to a carboxylicacid derivative that reacts readily with nucleophiles, generally muchmore readily than the underivatized carboxylic acid. Activatedcarboxylic acids include, for example, acid halides (such as acidchlorides), anhydrides, carbonates, and esters. Such esters includeimide esters, of the general form —(CO)O—N[(CO)—]₂; for example,N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters. Alsopreferred are imidazolyl esters and benzotriazole esters. Particularlypreferred are activated propionic acid or butanoic acid esters, asdescribed in co-owned U.S. Pat. No. 5,672,662. These include groups ofthe form —(CH₂)₂₋₃C(═O)O-Q, where Q is preferably selected fromN-succinimide, N-sulfosuccinimide, N-phthalimide, N-glutarimide,N-tetrahydrophthalimide, N-norbornene-2,3-dicarboximide, benzotriazole,7-azabenzotriazole, and imidazole.

Other preferred electrophilic groups include succinimidyl carbonate,maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate,p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyldisulfide.

These electrophilic groups are subject to reaction with nucleophiles,e.g., hydroxy, thio, or amino groups, to produce various bond types.Preferred for the present invention are reactions which favor formationof a hydrolytically stable linkage. For example, carboxylic acids andactivated derivatives thereof, which include orthoesters, succinimidylesters, imidazolyl esters, and benzotriazole esters, react with theabove types of nucleophiles to form esters, thioesters, and amides,respectively, of which amides are the most hydrolytically stable.Carbonates, including succinimidyl, imidazolyl, and benzotriazolecarbonates, react with amino groups to form carbamates. Isocyanates(R—N═C═O) react with hydroxyl or amino groups to form, respectively,carbamate (RNH—C(O)—OR′) or urea (RNH—C(O)—NHR′) linkages. Aldehydes,ketones, glyoxals, diones and their hydrates or alcohol adducts (i.e.,aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, andketal) are preferably reacted with amines, followed by reduction of theresulting imine, if desired, to provide an amine linkage (reductiveamination).

Several of the electrophilic functional groups include electrophilicdouble bonds to which nucleophilic groups, such as thiols, can be added,to form, for example, thioether bonds. These groups include maleimides,vinyl sulfones, vinyl pyridine, acrylates, methacrylates, andacrylamides. Other groups comprise leaving groups that can be displacedby a nucleophile; these include chloroethyl sulfone, pyridyl disulfides(which include a cleavable S—S bond), iodoacetamide, mesylate, tosylate,thiosulfonate, and tresylate. Epoxides react by ring opening by anucleophile, to form, for example, an ether or amine bond. Reactionsinvolving complementary reactive groups such as those noted above on theoligomer and the small molecule are utilized to prepare the conjugatesof the invention.

In some instances the foscarnet may not have a functional group suitedfor conjugation. In this instance, it is possible to modify (or“functionalize”) the “original” foscarnet so that it does have afunctional group suited for conjugation. For example, if the foscarnethas an amide group, but an amine group is desired, it is possible tomodify the amide group to an amine group by way of a Hofmannrearrangement, Curtius rearrangement (once the amide is converted to anazide) or Lossen rearrangement (once amide is concerted to hydroxamidefollowed by treatment with tolyene-2-sulfonyl chloride/base).

It is possible to prepare a conjugate of small molecule foscarnetbearing a carboxyl group wherein the carboxyl group-bearing smallmolecule foscarnet is coupled to an amino-terminated oligomeric ethyleneglycol, to provide a conjugate having an amide group covalently linkingthe small molecule foscarnet to the oligomer. This can be performed, forexample, by combining the carboxyl group-bearing small moleculefoscarnet with the amino-terminated oligomeric ethylene glycol in thepresence of a coupling reagent, (such as dicyclohexylcarbodiimide or“DCC”) in an anhydrous organic solvent.

Further, it is possible to prepare a conjugate of a small moleculefoscarnet bearing a hydroxyl group wherein the hydroxyl group-bearingsmall molecule foscarnet is coupled to an oligomeric ethylene glycolhalide to result in an ether (—O—) linked small molecule conjugate. Thiscan be performed, for example, by using sodium hydride to deprotonatethe hydroxyl group followed by reaction with a halide-terminatedoligomeric ethylene glycol.

Further, it is possible to prepare a conjugate of a small moleculefoscarnet moiety bearing a hydroxyl group wherein the hydroxylgroup-bearing small molecule foscarnet moiety is coupled to anoligomeric ethylene glycol bearing an haloformate group [e.g.,CH₃(OCH₂CH₂)_(n)OC(O)-halo, where halo is chloro, bromo, iodo] to resultin a carbonate [—O—C(O)—O—] linked small molecule conjugate. This can beperformed, for example, by combining a foscarnet moiety and anoligomeric ethylene glycol bearing a haloformate group in the presenceof a nucleophilic catalyst (such as 4-dimethylaminopyridine or “DMAP”)to thereby result in the corresponding carbonate-linked conjugate.

In another example, it is possible to prepare a conjugate of a smallmolecule foscarnet bearing a ketone group by first reducing the ketonegroup to form the corresponding hydroxyl group. Thereafter, the smallmolecule foscarnet now bearing a hydroxyl group can be coupled asdescribed herein.

In still another instance, it is possible to prepare a conjugate of asmall molecule foscarnet bearing an amine group. In one approach, theamine group-bearing small molecule foscarnet and an aldehyde-bearingoligomer are dissolved in a suitable buffer after which a suitablereducing agent (e.g., NaCNBH₃) is added. Following reduction, the resultis an amine linkage formed between the amine group of the aminegroup-containing small molecule foscarnet and the carbonyl carbon of thealdehyde-bearing oligomer.

In another approach for preparing a conjugate of a small moleculefoscarnet bearing an amine group, a carboxylic acid-bearing oligomer andthe amine group-bearing small molecule foscarnet are combined, typicallyin the presence of a coupling reagent (e.g., DCC). The result is anamide linkage formed between the amine group of the aminegroup-containing small molecule foscarnet and the carbonyl of thecarboxylic acid-bearing oligomer.

While it is believed that the full scope of the conjugates disclosedherein behave as described, an optimally sized oligomer can beidentified as follows.

First, an oligomer obtained from a monodisperse or bimodal water solubleoligomer is conjugated to the small molecule drug. Preferably, the drugis orally bioavailable, and on its own, exhibits a non-negligibleblood-brain barrier crossing rate. Next, the ability of the conjugate tocross the blood-brain barrier is determined using an appropriate modeland compared to that of the unmodified parent drug. If the results arefavorable, that is to say, if, for example, the rate of crossing issignificantly reduced, then the bioactivity of conjugate is furtherevaluated. Preferably, the compounds according to the invention maintaina significant degree of bioactivity relative to the parent drug, i.e.,greater than about 30% of the bioactivity of the parent drug, or evenmore preferably, greater than about 50% of the bioactivity of the parentdrug.

The above steps are repeated one or more times using oligomers of thesame monomer type but having a different number of subunits and theresults compared.

For each conjugate whose ability to cross the blood-brain barrier isreduced in comparison to the non-conjugated small molecule drug, itsoral bioavailability is then assessed. Based upon these results, that isto say, based upon the comparison of conjugates of oligomers of varyingsize to a given small molecule at a given position or location withinthe small molecule, it is possible to determine the size of the oligomermost effective in providing a conjugate having an optimal balancebetween reduction in biological membrane crossing, oral bioavailability,and bioactivity. The small size of the oligomers makes such screeningsfeasible and allows one to effectively tailor the properties of theresulting conjugate. By making small, incremental changes in oligomersize and utilizing an experimental design approach, one can effectivelyidentify a conjugate having a favorable balance of reduction inbiological membrane crossing rate, bioactivity, and oralbioavailability. In some instances, attachment of an oligomer asdescribed herein is effective to actually increase oral bioavailabilityof the drug.

For example, one of ordinary skill in the art, using routineexperimentation, can determine a best suited molecular size and linkagefor improving oral bioavailability by first preparing a series ofoligomers with different weights and functional groups and thenobtaining the necessary clearance profiles by administering theconjugates to a patient and taking periodic blood and/or urine sampling.Once a series of clearance profiles have been obtained for each testedconjugate, a suitable conjugate can be identified.

Animal models (rodents and dogs) can also be used to study oral drugtransport. In addition, non-in vivo methods include rodent everted gutexcised tissue and Caco-2 cell monolayer tissue-culture models. Thesemodels are useful in predicting oral drug bioavailability.

To determine whether the foscarnet or the conjugate of a foscarnet and awater-soluble non-peptidic polymer has activity as a foscarnettherapeutic, it is possible to test such a compound. The foscarnetcompounds may be tested using in vitro antiviral assays that have becomeroutine in pharmaceutical industry. The compounds according to theinvention may be subjected to biochemical tests.

Anti-Herpes Simplex Virus (HSV) Assay: A virus-induced cytopathiceffects (CPE)-inhibition assay procedure using Promega's Cell TiterAqueous One Solution (MTS, metabolic dye) is employed to evaluatecompounds for antiviral activity against herpes simplex virus type 1(strain HF) or herpes simplex virus type 2 (strain MS) in Vero cells.Vero cells are pre-grown in 96-well tissue culture plates usingDulbecco's Modified Eagle's Medium (DMEM) supplemented with 10%heat-inactivated fetal bovine serum (FBS), L Glutamine, penicillin, andstreptomycin. Antiviral assays are performed at a FBS concentration of2% and are designed to test six half-log dilutions of each compound intriplicate against the challenge virus in microtiter plate wellscontaining host cell monolayers. To each of the replicate cell culturesis added 50 μl of the test drug solution and 50 μl of virus suspension.Cell controls containing medium alone, virus-infected controlscontaining medium and virus, drug cytotoxicity controls containingmedium and each drug concentration, reagent controls containing culturemedium only (no cells), and drug colorimetric controls containing drugand medium (no cells) are run simultaneously with the test samples. Theplates are incubated at 37° C. in a humidified atmosphere containing 5%CO2 until maximum CPE is observed in the untreated virus controlcultures (Day 5).

MTS Staining for PBMC Viability to Measure Cytotoxicity: CPE inhibitionis determined by a dye (MTS) uptake procedure. This method measures cellviability and is based on the reduction of the tetrazolium-based MTS bymitochondrial enzymes of viable host cells to MTS formazan. MTS (10 μl)is added to each of the plate wells. The plates are incubated at 37° C.for 4 hours. The purple color of the MTS formazan is then measuredspectrophotometrically at 490/650 nm with a Molecular Devices Vmax orSpectraMaxPlus plate reader. The optical density (OD) value of eachculture is a function of the amount of formazan produced, which isproportional to the number of viable cells.

Data Analysis: Using an in-house computer program, IC50 (50%, inhibitionof virus replication), TC50 (50% cytotoxicity) and a therapeutic index(TI, TC50/IC50) are provided. Raw data for both antiviral activity andtoxicity with a graphical representation of the data are provided in aprintout summarizing the individual compound activity. Acyclovir wasevaluated in parallel as a relevant positive control compound in theanti-HSV assay.

Similarly, conjugates of the present invention may be tested for HIV-1antiviral cytoprotection assay using CEM-SS cells and thelaboratory-adapted HIV-1_(IIIB) strain. The activity may also be testedusing other viruses and suitable cell lines.

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, maltitol, lactitol, xylitol, sorbitol,myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines, fatty acidsand fatty esters; steroids, such as cholesterol; and chelating agents,such as EDTA, zinc and other such suitable cations.

Pharmaceutically acceptable acids or bases may be present as anexcipient in the preparation. Nonlimiting examples of acids that can beused include those acids selected from the group consisting ofhydrochloric acid, acetic acid, phosphoric acid, citric acid, malicacid, lactic acid, formic acid, trichloroacetic acid, nitric acid,perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, andcombinations thereof. Examples of suitable bases include, withoutlimitation, bases selected from the group consisting of sodiumhydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide,ammonium acetate, potassium acetate, sodium phosphate, potassiumphosphate, sodium citrate, sodium formate, sodium sulfate, potassiumsulfate, potassium fumerate, and combinations thereof.

The amount of the conjugate in the composition will vary depending on anumber of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container. Atherapeutically effective dose can be determined experimentally byrepeated administration of increasing amounts of the conjugate in orderto determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, excipients will be present in the composition in anamount of about 1% to about 99% by weight, preferably from about 5%-98%by weight, more preferably from about 15-95% by weight of the excipient,with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsand general teachings regarding pharmaceutical compositions aredescribed in “Remington: The Science & Practice of Pharmacy”, 19^(th)ed., Williams & Williams, (1995), the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H.,Handbook of Pharmaceutical Excipients, 3^(rd) Edition, AmericanPharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions can take any number of forms and theinvention is not limited in this regard. Exemplary preparations are mostpreferably in a form suitable for oral administration such as a tablet,caplet, capsule, gel cap, troche, dispersion, suspension, solution,elixir, syrup, lozenge, transdermal patch, spray, suppository, andpowder.

Oral dosage forms are preferred for those conjugates that are orallyactive, and include tablets, caplets, capsules, gel caps, suspensions,solutions, elixirs, and syrups, and can also comprise a plurality ofgranules, beads, powders or pellets that are optionally encapsulated.Such dosage forms are prepared using conventional methods known to thosein the field of pharmaceutical formulation and described in thepertinent texts.

Tablets and caplets, for example, can be manufactured using standardtablet processing procedures and equipment. Direct compression andgranulation techniques are preferred when preparing tablets or capletscontaining the conjugates described herein. In addition to theconjugate, the tablets and caplets will generally contain inactive,pharmaceutically acceptable carrier materials such as binders,lubricants, disintegrants, fillers, stabilizers, surfactants, coloringagents, flow agents, and the like. Binders are used to impart cohesivequalities to a tablet, and thus ensure that the tablet remains intact.Suitable binder materials include, but are not limited to, starch(including corn starch and pregelatinized starch), gelatin, sugars(including sucrose, glucose, dextrose and lactose), polyethylene glycol,waxes, and natural and synthetic gums, e.g., acacia sodium alginate,polyvinylpyrrolidone, cellulosic polymers (including hydroxypropylcellulose, hydroxypropyl methylcellulose, methyl cellulose,microcrystalline cellulose, ethyl cellulose, hydroxyethylcellulose, andthe like), and Veegum. Lubricants are used to facilitate tabletmanufacture, promoting powder flow and preventing particle capping(i.e., particle breakage) when pressure is relieved. Useful lubricantsare magnesium stearate, calcium stearate, and stearic acid.Disintegrants are used to facilitate disintegration of the tablet, andare generally starches, clays, celluloses, algins, gums, or crosslinkedpolymers. Fillers include, for example, materials such as silicondioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose,and microcrystalline cellulose, as well as soluble materials such asmannitol, urea, sucrose, lactose, dextrose, sodium chloride, andsorbitol. Stabilizers, as well known in the art, are used to inhibit orretard drug decomposition reactions that include, by way of example,oxidative reactions.

Capsules are also preferred oral dosage forms, in which case theconjugate-containing composition can be encapsulated in the form of aliquid or gel (e.g., in the case of a gel cap) or solid (includingparticulates such as granules, beads, powders or pellets). Suitablecapsules include hard and soft capsules, and are generally made ofgelatin, starch, or a cellulosic material. Two-piece hard gelatincapsules are preferably sealed, such as with gelatin bands or the like.

Included are parenteral formulations in the substantially dry form(typically as a lyophilizate or precipitate, which can be in the form ofa powder or cake), as well as formulations prepared for injection, whichare typically liquid and requires the step of reconstituting the dryform of parenteral formulation. Examples of suitable diluents forreconstituting solid compositions prior to injection includebacteriostatic water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline, sterile water,deionized water, and combinations thereof.

In some cases, compositions intended for parenteral administration cantake the form of nonaqueous solutions, suspensions, or emulsions, eachtypically being sterile. Examples of nonaqueous solvents or vehicles arepropylene glycol, polyethylene glycol, vegetable oils, such as olive oiland corn oil, gelatin, and injectable organic esters such as ethyloleate.

The parenteral formulations described herein can also contain adjuvantssuch as preserving, wetting, emulsifying, and dispersing agents. Theformulations are rendered sterile by incorporation of a sterilizingagent, filtration through a bacteria-retaining filter, irradiation, orheat.

The conjugate can also be administered through the skin usingconventional transdermal patch or other transdermal delivery system,wherein the conjugate is contained within a laminated structure thatserves as a drug delivery device to be affixed to the skin. In such astructure, the conjugate is contained in a layer, or “reservoir,”underlying an upper backing layer. The laminated structure can contain asingle reservoir, or it can contain multiple reservoirs.

The conjugate can also be formulated into a suppository for rectaladministration. With respect to suppositories, the conjugate is mixedwith a suppository base material which is (e.g., an excipient thatremains solid at room temperature but softens, melts or dissolves atbody temperature) such as coca butter (theobroma oil), polyethyleneglycols, glycerinated gelatin, fatty acids, and combinations thereof.Suppositories can be prepared by, for example, performing the followingsteps (not necessarily in the order presented): melting the suppositorybase material to form a melt; incorporating the conjugate (either beforeor after melting of the suppository base material); pouring the meltinto a mold; cooling the melt (e.g., placing the melt-containing mold ina room temperature environment) to thereby form suppositories; andremoving the suppositories from the mold.

In some embodiments of the invention, the compositions comprising theconjugates may further be incorporated into a suitable delivery vehicle.Such delivery vehicles may provide controlled and/or continuous releaseof the conjugates and may also serve as a targeting moiety. Non-limitingexamples of delivery vehicles include, adjuvants, synthetic adjuvants,microcapsules, microparticles, liposomes, and yeast cell wall particles.Yeast cells walls may be variously processed to selectively removeprotein component, glucan, or mannan layers, and are referred to aswhole glucan particles (WGP), yeast beta-glucan mannan particles (YGMP),yeast glucan particles (YGP), \Rhodotorula yeast cell particles (YCP).Yeast cells such as S. cerevisiae and Rhodotorula sp. are preferred;however, any yeast cell may be used. These yeast cells exhibit differentproperties in terms of hydrodynamic volume and also differ in the targetorgan where they may release their contents. The methods of manufactureand characterization of these particles are described in U.S. Pat. Nos.5,741,495; 4,810,646; 4,992,540; 5,028,703; 5,607,677, and U.S. PatentApplications Nos. 2005/0281781, and 2008/0044438.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with the conjugate. The method comprisesadministering, generally orally, a therapeutically effective amount ofthe conjugate (preferably provided as part of a pharmaceuticalpreparation). Other modes of administration are also contemplated, suchas pulmonary, nasal, buccal, rectal, sublingual, transdermal, andparenteral. As used herein, the term “parenteral” includes subcutaneous,intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal,and intramuscular injection, as well as infusion injections.

In instances where parenteral administration is utilized, it may benecessary to employ somewhat bigger oligomers than those describedpreviously, with molecular weights ranging from about 500 to 30K Daltons(e.g., having molecular weights of about 500, 1000, 2000, 2500, 3000,5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).

The method of administering may be used to treat any condition that canbe remedied or prevented by administration of the particular conjugate.Those of ordinary skill in the art appreciate which conditions aspecific conjugate can effectively treat. The actual dose to beadministered will vary depend upon the age, weight, and generalcondition of the subject as well as the severity of the condition beingtreated, the judgment of the health care professional, and conjugatebeing administered. Therapeutically effective amounts are known to thoseskilled in the art and/or are described in the pertinent reference textsand literature. Generally, a therapeutically effective amount will rangefrom about 0.001 mg to 1000 mg, preferably in doses from 0.01 mg/day to750 mg/day, and more preferably in doses from 0.10 mg/day to 500 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

All articles, books, patents, patent publications and other publicationsreferenced herein are incorporated by reference in their entireties. Inthe event of an inconsistency between the teachings of thisspecification and the art incorporated by reference, the meaning of theteachings in this specification shall prevail.

EXPERIMENTAL

It is to be understood that while the invention has been described inconjunction with certain preferred and specific embodiments, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All non-PEG chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated. The preparation ofPEG-mers is described in, for example, U.S. Patent ApplicationPublication No. 2005/0136031.

All ¹H NMR (nuclear magnetic resonance) data was generated by an NMRspectrometer. A list of certain compounds as well as the source of thecompounds is provided below.

Example 1 Synthesis of mPEG_(n)-Foscarnet Conjugates

Synthesis of mPEG_(n)-OCO—Cl (n=3, 7): The chloroform (˜6 mL) solutionof mPEG_(n)-OH (1.0 g, 2.94 mmol) and TEA (0.36 g, 3.56 mmol) was cooledto −10° C. under nitrogen. After stirring for 5 min, triphosgene (0.65g, 2.20 mmol) was added and the solution was allowed to stir for anadditional 5 min at −10° C. The reaction mixture was slowly warmed up toroom temperature and stirred overnight. The solvent was removed underreduced pressure and then Et₂O (˜50 mL) was added. After stirring for 15min, the white precipitate was collected by filtration and the filtratewas concentrated to give a clear liquid (˜1.2 g, ˜98% yield). ¹H NMRconfirmed that it was the desired mPEG_(n)-OCO—Cl (˜96% conversion).

Microwave synthesis of mPEG_(n)-OCO—P(O)(OSiMe₃)₂ (n=3, 7): Animmiscible mixture of mPEG_(n)-OCO—Cl (1.12 g, 2.78 mmol) andtris(trimethylsilyl)phosphate (1.24 g, 4.17 mmol) without any solventwas irradiated in a microwave at 100° C. for 10 min at which point ahomogenous solution was formed. The excess tris(trimethylsilyl)phosphatewas then removed under reduced pressure at 80° C. The desiredmPEG_(n)-OCO—P(O)(OSiMe₃)₂ was obtained as a clear liquid (˜1.66 g, ˜98%yield). ¹H NMR indicated that all mPEG_(n)-OCO—Cl was converted into theproduct and almost all excess tris(trimethylsilyl)phosphate was removed.

Synthesis of mPEG_(n)-foscarnet (n=3, 7): To a DCM (˜5 mL) solution ofmPEG_(n)-OCO—P(O)(OSiMe₃)₂ (200 mg, 0.34 mmol) was added 1.35 mL of 0.5M NaOCH₃ methanol solution and the solution was stirred at roomtemperature for 10 min. The solvent was removed under reduced pressureand dried in vacuum, to a white waxy solid (160 mg, ˜95% yield). ¹H NMR(D₂O) confirmation of the desired mPEG_(n)-OCO—P(O)(ONa)₂ was obtained.

II. Synthesis of mPEG_(n)-Foscarnet-Lipid Conjugates

Synthesis of mPEG₃-OCO—Cl: To a DCM solution of mPEG₃-OH (˜3.0 g, ˜18.3mmol) and TEA (˜2.2 g, ˜21.9 mmol) was added triphosgene (˜4.08 g, ˜13.7mmol). The reaction solution was stirred at room temperature undernitrogen overnight. ¹H NMR (CDCl₃) showed that the reaction wascomplete. All the solvents were removed under reduced pressure and tothe resulting white residue was added diethyl ether (˜50 mL). Afterstirring the mixture at room temperature for 10 min, the white solid(TEA HCl salt) was obtained by filtration. The filtrate was concentratedand dried in vacuo to give a light yellow liquid (˜3.6 g, ˜87% isolatedyield). ¹H NMR (CDCl₃) indicated that the desired mPEG₃-OCO—Cl wasobtained (purity>95%).

Microwave synthesis of mPEG₃-OCO—P(O)(OSiMe₃)₂: A mixture ofmPEG₃-OCO—Cl (˜520 mg, ˜2.29 mmol) and P(OSiMe₃)₃ (˜698 mg, ˜2.34 mmol)was irradiated with a microwave at 100° C. for 10 min. ¹H NMR showedthat the desired mPEG₃-OCO—P(O)(OSiMe₃)₂ was formed as a clear viscousliquid (˜91%). The product mixture was not purified and used as such innext step.

Synthesis of mPEG₃-OCO—P(O)(OH)₂: mPEG₃-OCO—P(O)(OSiMe₃)₂ (˜2.29 mmol)was dissolved in 1:1 pyridine/CH₃OH solution, the reaction mixture wasstirred at room temperature for 5 min. The solvents were removed underreduced pressure and further dried in vacuo. The desiredmPEG₃-OCO—P(O)(OH)₂ was obtained as a clear sticky liquid and was notpurified and used as such in next step.

Synthesis of mPEG₃-OCO—P(O)(OH)[O—(CH₂)₆CH₃]: To a pyridine (˜8 mL)solution of mPEG₃-OCO—P(O)(OH)₂ (˜2.29 mmol) was added 1-heptanol (˜293mg, ˜2.52 mmol), then added DCC (˜1.42 g, ˜6.87 mmol). The reactionmixture was stirred at room temperature under nitrogen overnight. Whiteprecipitates were formed gradually. The white DCU solid was removed byfiltration; the resulting filtrate was evaporated under reduced pressureto give a mixture of liquid and solid. The product mixture was placed ona 20×300 mm silica gel column, the column was first eluted with pureDCM, then gradually from 1:20 CH₃OH/DCM to 1:1 CH₃OH/DCM. Each fractioncollected was evaporated and measured by proton NMR (d₄-methanol). ˜100mg of the desired mPEG₃-OCO—P(O)(OH)[O—(CH₂)₆CH₃] was obtained. ¹H NMR(d₄-methanol) showed exactly one heptanol molecule was coupled tomPEG₃-OCO-Foscarnet. LC-MS further confirmed themPEG₃-OCO—P(O)(OH)[O—(CH₂)₆CH₃] structure, Calc Mass: 370.4; Found Mass:370.9

III. Alternate Synthesis of mPEG_(n)-Foscarnet-Lipid Conjugates

IV. Synthesis of1-m-PEG3-2-MeO-Glycerol-Fosacrnet-O-2-MeO-Glycerol-1-Octadecyl and1-m-PEG3-2-EtO-Glycerol-Fosacrnet-O-2-EtO-Glyecrol-1-Octadecyl

Synthesis of 1-m-PEG3-2-MeO-Glycerol-Chloroformate: To a DCM solution of1-m-PEG3-2-MeO-glycerol (˜190 mg; ˜0.753 mmol) and TEA (˜84 mg, ˜0.828mmol) in an ice-bath at 0° C. was added triphosgene solid (˜224 mg,˜0.753 mmol). The reaction solution was stirred at room temperatureunder nitrogen for 4 h. ¹H NMR (CDCl₃) showed that the reaction wascomplete. Solvents were removed with a rotary evaporator; the whiteresidue was added to diethyl ether (˜50 mL). After stirring the mixtureat room temperature for 10 min, white solid (TEA HCl salt) was filteredoff. The filtrate was evaporated and dried in vacuum to give a lightyellow liquid (˜220 mg, ˜99% isolated yield). ¹H NMR (CDCl₃) indicatedthat the purity of 1-m-PEG3-2-MeO-glycerol-OCO—Cl was >96%.

Microwave synthesis of 1-m-PEG3-2-MeO-glycerol-OCO—P(O)(OSiMe₃)₂: Amixture of 1-m-PEG3-2-MeO-glycerol-OCO—Cl (˜220 mg, ˜0.75 mmol) andP(OSiMe₃)₃ (˜224 mg, ˜0.75 mmol) was dissolved in dichloromethane (˜3mL), then stirred in vacuum to ensure sufficient mixing. The residue wasirradiated with microwave at 100° C. for 15 min. ¹H NMR showed that1-m-PEG3-2-MeO-glycerol-OCO—P(O)(OSiMe₃)₂ was formed in ˜91% as a clearviscous liquid. The product mixture was not purified and used as such innext step reaction.

Synthesis of 1-m-PEG3-2-MeO-glycerol-OCO—P(O)(OH)₂:1-m-PEG3-2-MeO-glycerol-OCO—P(O)(OSiMe₃)₂ (˜0.75 mmol) was dissolved in2:1 pyridine/CH₃OH, the solution was stirred at room temperature forfive min. All solvents were then removed with a rotary evaporator. Afterfurther drying in vacuum, 1-m-PEG3-2-MeO-glycerol-OCO—P(O)(OH)₂ wasobtained as a clear sticky liquid. The product was not purified and usedas such in next step reaction.

Synthesis of1-m-PEG3-2-MeO-glycerol-OCO—P(O)(OH)(O-2-MeO-glycerol-1-Octadecyl): Thetoluene (˜25 mL) solution of 1-m-PEG3-2-MeO-glycerol-OCO—P(O)(OH)₂ and1-Octadecyl-2-MeO-glycerol (˜300 mg, ˜0.84 mmol) was azeotropicallydistilled under vacuum. The residue was redissolved in anhydrouspyridine (˜10 mL), then added DCC (˜463 mg, ˜2.25 mmol) in CH₂Cl₂ (˜1mL) solution. The reaction mixture was stirred at room temperature undernitrogen overnight (˜18 h). White DCU precipitates were formedgradually. All the solvents were evaporated with a rotary evaporator togive a semi-solid. The residues were dissolved in CH₂Cl₂, after filteredoff the white precipitates, the light yellow filtrate was placed on a20×400 mm silica gel column prepared in 1:50 CH₃OH/CH₂Cl₂, the columnwas first eluted with 1:50 CH₃OH/CH₂Cl₂, then 1:10 CH₃OH/CH₂Cl₂, then1:3 CH₃OH/CH₂Cl₂. The product quickly came out when 1:3 CH₃OH/CH₂Cl₂mixture solvent was used. ˜60 mg of1-m-PEG3-2-MeO-glycerol-OCO—P(O)(OH)(O-2-MeO-glycerol-1-Octadecyl) wasobtained as a sticky liquid (˜0.086 mmol, ˜11% isolated yield). TLC onlyshowed one spot (in iodine). ¹H NMR (d₄-methanol) confirmed the product.MALDI-MS further confirmed the desired product structure, Calc Mass:700.9; Found Mass: 700.7.

Synthesis of 1-m-PEG3-2-EtO-Glycerol-Chloroformate: To a DCM solution of1-m-PEG3-2-EtO-glycerol (˜200 mg, ˜0.75 mmol) and TEA (˜76 mg, ˜0.75mmol) in an acetone-bath at −20° C. was added triphosgene solid (˜223mg, ˜0.75 mmol). The reaction solution was slowly warmed to roomtemperature under nitrogen. After 4 h, ¹H NMR (CDCl₃) showed that thereaction was complete. All the solvents were removed with a rotaryevaporator; the white residue was added to diethyl ether (˜50 mL). Afterstirring the mixture at room temperature for 10 min, white solid (TEAHCl salt) was filtered off. The filtrate was evaporated and dried invacuum to give a clear liquid (˜210 mg, ˜85% isolated yield). ¹H NMR(CDCl₃) indicated that the purity of 1-m-PEG3-2-EtO-glycerol-OCO—Cl was>96%.

Microwave synthesis of 1-m-PEG3-2-EtO-glycerol-OCO—P(O)(OSiMe₃)₂ Amixture of 1-m-PEG3-2-EtO-glycerol-OCO—Cl (˜210 mg, ˜0.64 mmol) andP(OSiMe₃)₃ (˜224 mg, ˜0.75 mmol) was dissolved in dichloromethane (˜3mL), then dried in vacuum to ensure sufficient mixing. The residue wasirradiated with microwave at 100° C. for 15 min. ¹H NMR showed that1-m-PEG3-2-EtO-glycerol-OCO—P(O)(OSiMe₃)₂ was formed. The productmixture was not purified and used as such in next step reaction.

Synthesis of 1-m-PEG3-2-EtO-glycerol-OCO—P(O)(OH)₂:1-m-PEG3-2-EtO-glycerol-OCO—P(O)(OSiMe₃)₂ (˜0.64 mmol) was dissolved in˜3 mL of 2:1 pyridine/CH₃OH, the solution was stirred at roomtemperature for 5 min. All solvents were then removed with a rotaryevaporator. After further dried in vacuum,1-m-PEG3-2-EtO-glycerol-OCO—P(O)(OH)₂ was obtained as a clear stickyliquid. The product was not purified and used as such in next stepreaction.

Synthesis of1-m-PEG3-2-EtO-glycerol-OCO—P(O)(OH)(O-2-EtO-glycerol-1-Octadecyl): Thetoluene (˜30 mL) solution of 1-m-PEG3-2-EtO-glycerol-OCO—P(O)(OH)₂ (fromstep 7) and 1-Octadecyl-2-EtO-glycerol (˜240 mg, ˜0.64 mmol) wasazeotropically distilled under vacuum. The residue was redissolved inanhydrous pyridine (˜8 mL), then added DCC (˜463 mg, ˜2.25 mmol) inCH₂Cl₂ (˜1 mL) solution. The reaction mixture was stirred at roomtemperature under nitrogen overnight (˜16 h). White DCU precipitateswere formed gradually. All the solvents were evaporated with a rotaryevaporator to give a semi-solid. The residues were dissolved in CH₂Cl₂,after filtered off the white precipitates, the light yellow filtrate wasplaced on a 20×400 mm silica gel column prepared in 1:50 CH₃OH/CH₂Cl₂,the column was first eluted with 1:50 CH₃OH/CH₂Cl₂, then 1:10CH₃OH/CH₂Cl₂, then 1:3 CH₃OH/CH₂Cl₂. The product quickly came out when1:3 CH₃OH/CH₂Cl₂ mixture solvent was used. ˜83 mg of1-m-PEG3-2-EtO-glycerol-OCO—P(O)(OH)(O-2-EtO-glycerol-1-Octadecyl) wasobtained as a sticky liquid (˜0.11 mmol, ˜18% isolated yield). TLC onlyshowed one spot (in iodine). ¹H NMR and ³¹P NMR (˜5.33 ppm, d₄-methanol)confirmed the product. MALDI-MS further confirmed the desired productstructure, Calc Mass: 728; Found Mass: 728.

Analytical Methods: NMR: The ¹H NMR and ³¹P NMR (10% phosphoric acid asreference) measurements were performed on a Bruker 300 NMR spectrometer.Microwave synthesis: All microwave synthesis was performed on aMilestone MicroSYNTH Labstation or CEM microwave station.

Conjugates of PEG-foscarnet are also synthesized using glyceraldehyde.In this instant, glyceraldehydes would be reductively deaminated to forman N-linked oligomer conjugate.

Example 2 Anti-HSV and Anti-HIV Assays

Foscarnet, mPEG₃-OCO—P(O)(ONa)₂, and mPEG₃-OCO—P(O)(OH)[O—(CH₂)₆CH₃]were tested for their ability to inhibit viral cytopethic effects andalso for their effects on cell viability in Vero cells, and CEM-SS cellsas described above.

Anti-Herpes Simplex Virus (HSV) Assay: A virus-induced cytopathiceffects (CPE)-inhibition assay procedure using Promega's Cell TiterAqueous One Solution (MTS, metabolic dye) is employed to evaluatecompounds for antiviral activity against herpes simplex virus type 1(strain HF) or herpes simplex virus type 2 (strain MS) in Vero cells.Vero cells are pre-grown in 96-well tissue culture plates usingDulbecco's Modified Eagle's Medium (DMEM) supplemented with 10%heat-inactivated fetal bovine serum (FBS), L Glutamine, penicillin, andstreptomycin. Antiviral assays are performed at a FBS concentration of2% and are designed to test six half-log dilutions of each compound intriplicate against the challenge virus in microtiter plate wellscontaining host cell monolayers. To each of the replicate cell culturesis added 50 μl of the test drug solution and 50 μl of virus suspension.Cell controls containing medium alone, virus-infected controlscontaining medium and virus, drug cytotoxicity controls containingmedium and each drug concentration, reagent controls containing culturemedium only (no cells), and drug colorimetric controls containing drugand medium (no cells) are run simultaneously with the test samples. Theplates are incubated at 37° C. in a humidified atmosphere containing 5%CO2 until maximum CPE is observed in the untreated virus controlcultures (Day 5).

MTS Staining for PBMC Viability to Measure Cytotoxicity: CPE inhibitionis determined by a dye (MTS) uptake procedure. This method measures cellviability and is based on the reduction of the tetrazolium-based MTS bymitochondrial enzymes of viable host cells to MTS formazan. MTS (10 μl)is added to each of the plate wells. The plates are incubated at 37° C.for 4 hours. The purple color of the MTS formazan is then measuredspectrophotometrically at 490/650 nm with a Molecular Devices Vmax orSpectraMaxPlus plate reader. The optical density (OD) value of eachculture is a function of the amount of formazan produced, which isproportional to the number of viable cells.

Data Analysis: Using an in-house computer program, IC50 (50%, inhibitionof virus replication), TC50 (50% cytotoxicity) and a therapeutic index(TI, TC50/IC50) are provided. Raw data for both antiviral activity andtoxicity with a graphical representation of the data are provided in aprintout summarizing the individual compound activity. Acyclovir wasevaluated in parallel as a relevant positive control compound in theanti-HSV assay.

Cell Culture (ATCC CCL-81): Vero cells (Kidney, African green monkey,Cercopithecus aethiops) were obtained from the American Type CultureCollection (ATCC, Rockville, Md.) and are grown in Dulbecco's ModifiedEagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS),2.0 mM L-Glutamine, 100 units/ml Penicillin and 100 ug/ml Streptomycin(“growth medium”). Cells are sub-cultured twice a week at a split ratioof 1:10 using standard cell culture techniques.

HSV-1 Strain HF and HSV-2 Strain MS were obtained from the ATCC. Virusstocks were prepared by infecting approximately 80% confluent monolayersof Vero cells at a minimal multiplicity of infection in growth mediumcontaining a reduced FBS concentration (2%). Monolayers were incubatedat 37° C. and 5% CO2 until 90-95% viral cytopathic effect (CPE) wasobserved (4-5 days). Culture medium was then collected from the cells,centrifuged at low speed to remove cellular debris, aliquoted in 1 mlvolumes and frozen at −80° C. as stock virus. Optimal amounts of inputvirus for use in the CPE assay were determined by using serial dilutionswithin the assay and selecting the minimum amount of virus necessary toproduce maximum CPE within five days of incubation.

Foscarnet, mPEG₃-OCO—P(O)(ONa)₂, and mPEG₃-OCO—P(O)(OH)[O—(CH₂)₆CH₃] didnot exhibit CPE up to the highest tested concentration of 400 mM.

CEM-SS cells were passaged in T-75 flasks prior to use in the antiviralassay. On the day preceding the assay, the cells were split 1:2 toassure they were in an exponential growth phase at the time ofinfection. Total cell and viability quantification was performed using ahemacytometer and trypan blue exclusion. Cell viability was greater than95% for the cells to be utilized in the assay. The cells werere-suspended at 5×104 cells/ml in tissue Culture medium and added to thedrug-containing microtiter plates in a volume of 50 μl.

The virus used for these tests was the lymphocytropic virus strain HIV-1IIIB. This virus was obtained from the NIH AIDS Research and ReferenceReagent Program and was grown in CEM-SS cells for the production ofstock virus pools. For each assay, a pre-titered aliquot of virus wasremoved from the freezer and allowed to thaw slowly to room temperaturein a biological safety cabinet. The virus was re-suspended and dilutedinto tissue culture medium such that the amount of virus added to eachwell in a volume of 50 μl was the amount determined to giveapproximately 90% cell killing at 6 days post-infection. TCID50calculations by endpoint titration in CEM-SS cells indicated that themultiplicity of infection of these assays was approximately 0.01.

Foscarnet exhibited IC₅₀ of 29.6 mM, while mPEG₃-OCO—P(O)(ONa)₂, andmPEG₃-OCO—P(O)(OH)[O—(CH₂)₆CH₃] did not exhibit CPE up to the highesttested concentration of 400 mM.

What is claimed is:
 1. A pharmaceutical composition comprising acompound and a pharmaceutically acceptable excipient, wherein thecompound has the following structure:

wherein: X is a spacer moiety of less than about five atoms; and POLY isa water-soluble, non-peptidic oligomer, wherein the water-soluble,non-peptidic oligomer comprises 3 to 30 monomers of an alkylene oxide.2. The pharmaceutical composition of claim 1, wherein the alkylene oxideis an ethylene oxide.
 3. The pharmaceutical composition of claim 1,wherein the alkylene oxide includes an alkoxy or hydroxy end-cappingmoiety.
 4. The pharmaceutical composition of claim 1, wherein the spacermoiety is a stable linkage.
 5. The pharmaceutical composition of claim1, wherein the spacer moiety is a degradable linkage.
 6. The compound ofclaim 1, wherein the spacer moiety is an ester linkage.